PROJECT SUMMARY/ABSTRACT The vector-borne spirochetes that cause relapsing fever are transmitted to humans by either soft ticks or human body lice. Despite identification of the etiological agents of relapsing fever over 100 years ago, very little information exists regarding their pathogenesis. Although relapsing fever is more common in developing countries, tick-borne relapsing fever (TBRF) occurs in areas of the U.S. where Ornithodoros species of soft ticks, the vectors for TBRF spirochetes, are endemic. During their natural enzootic cycle, vector-borne spirochetes exist in two distinct niches found within the arthropod vector and the vertebrate. It is well established that Lyme disease spirochetes must undergo significant changes in global gene expression to allow them to adapt to these two diverse environments, and a great deal of information exists regarding the regulatory networks that control this bacterial adaptive response. However, the correlate that occurs in TBRF spirochetes to facilitate vector-mediated transmission and mammalian infection remains undefined. Our overall goal is to expand our knowledge of the pathogenesis of TBRF spirochetes and to identify virulence determinants (e.g., virulence factors and regulatory components) required during mammalian infection. We hypothesize that TBRF spirochetes must sense environmental temperature and utilize this stimulus to control expression of genes essential for mammalian infection and vector transmission. In preliminary experiments, we first defined the global temperature-dependent gene response that occurs in Borrelia turicatae, one of the major Borrelia species responsible for TBRF in the U.S., and then used these data to establish a set of genes that will be characterized during the course of studies in this project. Aim 1 seeks to identify cis- and trans-regulatory elements responsible for controlling the expression of temperature-responsive genes in B. turicatae. In Aim 2, we will use targeted mutagenesis to inactivate individual temperature-responsive genes in B. turicatae and identify bacterial factors required for infection and pathogenesis. These aims will provide critical knowledge regarding differential gene regulation occurring in TBRF spirochetes during transmission and infection, and identify virulence determinants required by the bacteria to cause disease. This research is aligned with the COBRE program's focus on microbial pathogenesis by defining molecular determinants of TBRF spirochetes that contribute to adaptation and interaction within the mammalian host. Regulators and virulence factors identified in this project represent potential targets against which future therapeutic interventions and/or diagnostics for TBRF could be developed. The further characterization of these virulence determinants will be the focus of future R01 grant proposals.